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Dietary conjugated α-linolenic acid (CLNA) did not improve glucose tolerance in a

neonatal pig model

--Manuscript

Draft--Manuscript Number:

Full Title: Dietary conjugated α-linolenic acid (CLNA) did not improve glucose tolerance in a neonatal pig model

Article Type: Original Contribution

Keywords: conjugated linolenic acid; n-3 fatty acid; insulin resistance; pig Corresponding Author: Christian-Alexandre Castellano, Ph.D.

Research Center on Aging - Université de Sherbrooke Sherbrooke, QC CANADA

Corresponding Author Secondary Information:

Corresponding Author's Institution: Research Center on Aging - Université de Sherbrooke Corresponding Author's Secondary

Institution:

First Author: Christian-Alexandre Castellano, Ph.D. First Author Secondary Information:

Order of Authors: Christian-Alexandre Castellano, Ph.D. Jean-Patrice Baillargeon, M.D Mélanie Plourde, Ph.D. Sandie I. Briand Paul Anger, Ph.D. Alain Giguère J. Jacques Matte, Ph.D. Order of Authors Secondary Information:

Abstract: Purpose: There is an increased interest in the benefits of conjugated α-linolenic acid (CLNA) on obesity-related complications such as insulin resistance and diabetes. The aim of the study was to investigate whether a 1% dietary supplementation of mono-CLNA isomers (c9-t11-c15-18:3 + c9-t13-c15-18:3) improved glucose and lipid metabolism in neonatal pigs. Methods: Since mono-CLNA isomers combine one conjugated two-double bond system with an n-3 polyunsaturated fatty acid (PUFA) structure, the experimental protocol was designed to isolate the dietary structural characteristics of the molecules by comparing a CLNA diet with three other dietary fats: 1) conjugated linoleic acid (c9-t11-18:2 + t10-c12-18:2; CLA), 2) non-conjugated n-3 PUFA and 3) n-6 PUFA. Thirty-two piglets weaned at 3 weeks of age were distributed into the four dietary groups. Diets were isoenergetic and food intake was controlled by a gastric tube. After 2 weeks of supplementation, gastro-enteral (OGTT) and parenteral (IVGTT) glucose tolerance tests were conducted. Results: Dietary supplementation with mono-CLNA did not modify body weight/fat or blood lipid profiles (p>0.82 and p>0.57, respectively) compared with other dietary groups. Plasma glucose, insulin and C-peptide responses to OGTT and IVGTT in the CLNA group was not different from the three other dietary groups (p>0.18 and p>0.15, respectively). Compared to the non-conjugated n-3 PUFA diet, CLNA-fed animals had decreased liver composition in three n-3 fatty acids (18:3n-3; 20:3n-3; 22:5n-3) (p<0.001). Conclusions: These results suggest that providing 1% mono-CLNA is not effective in improving insulin sensitivity in neonatal pigs.

Suggested Reviewers: Hélène Poirier

h.poirier@agrosupdijon.fr

(2)

Darshan S. Kelley

darshan.kelley@ars.usda.gov

(3)

CLNA and glucose tolerance in neonatal pigs European Journal of Nutrition – v2013-06-21

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Title: Dietary conjugated α-linolenic acid (CLNA) did not improve glucose tolerance in a neonatal pig model

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Running title: CLNA and glucose tolerance in neonatal pigs

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Christian-Alexandre Castellano1,2*, Jean-Patrice Baillargeon3, Mélanie Plourde1,3, Sandie I. Briand4,5, Paul Angers6, Alain

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Giguère7 and J. Jacques Matte7

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1Research Center on Aging, Health and Social Sciences Center, Geriatrics Institute, Sherbrooke, QC, Canada

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2Department Physiology and Biophysics, Université de Sherbrooke, Sherbrooke, QC, Canada

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3Department of Medicine, Division of Endocrinology, Université de Sherbrooke, Sherbrooke, QC, Canada

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4Naturia Inc., Sherbrooke, QC, Canada

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5Institut national de santé publique du Québec, Montréal, QC, Canada

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6Department of Food Sciences and Nutrition, Faculty of Agriculture and Agri-Food Sciences, Université Laval, QC,

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Canada, G1K 7P4.

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Dairy and Swine Research and Development Centre, Agriculture and Agri-Food Canada, Sherbrooke, QC, Canada

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*Corresponding author:

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Research Center on Aging, 1036 Belvedere South, Sherbrooke, (QC, Canada) J1H 4C4.

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Tel: 1-819-780-2220 - Fax: 1-819-829-7141

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E-mail: Alexandre.Castellano@USherbrooke.ca

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Abstract

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Purpose: There is an increased interest in the benefits of conjugated α-linolenic acid (CLNA) on obesity-related

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complications such as insulin resistance and diabetes. The aim of the study was to investigate whether a 1% dietary

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supplementation of mono-CLNA isomers (c9-t11-c15-18:3 + c9-t13-c15-18:3) improved glucose and lipid metabolism in

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neonatal pigs. Methods: Since mono-CLNA isomers combine one conjugated two-double bond system with an n-3

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polyunsaturated fatty acid (PUFA) structure, the experimental protocol was designed to isolate the dietary structural

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characteristics of the molecules by comparing a CLNA diet with three other dietary fats: 1) conjugated linoleic acid

(c9-t11-32

18:2 + t10-c12-18:2; CLA), 2) non-conjugated n-3 PUFA and 3) n-6 PUFA. Thirty-two piglets weaned at 3 weeks of age

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were distributed into the four dietary groups. Diets were isoenergetic and food intake was controlled by a gastric tube. After

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2 weeks of supplementation, gastro-enteral (OGTT) and parenteral (IVGTT) glucose tolerance tests were conducted.

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Results: Dietary supplementation with mono-CLNA did not modify body weight/fat or blood lipid profiles (p>0.82 and

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p>0.57, respectively) compared with other dietary groups. Plasma glucose, insulin and C-peptide responses to OGTT and

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IVGTT in the CLNA group was not different from the three other dietary groups (p>0.18 and p>0.15, respectively).

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Compared to the non-conjugated n-3 PUFA diet, CLNA-fed animals had decreased liver composition in three n-3 fatty

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acids (18:3n-3; 20:3n-3; 22:5n-3) (p<0.001). Conclusions: These results suggest that providing 1% mono-CLNA is not

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effective in improving insulin sensitivity in neonatal pigs.

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Key words: conjugated linolenic acid: n-3 fatty acid: insulin resistance: pig

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Introduction

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Conjugated fatty acids refer to a set of positional and geometric isomers of polyunsaturated fatty acids (PUFA) with

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conjugated double bonds. Conjugated linoleic acids (CLA) were first identified in the late eighties by Pariza et al. [1]. Since

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then, consumption of CLA was associated to weight loss [2] and improving of insulin sensitivity [3]. Another group of

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conjugated fatty acids recently received more attention because it combined an n-3 and a conjugated double bond:

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conjugated α-linolenic acids (CLNA) [4]. CLNA isomers are naturally present in plant seeds (di-CLNA) and in dairy

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products (mono-CLNA). Mono- and di-CLNA differ by their conjugated double-bond system: mono-CLNA have a single

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conjugated double-bond system at the n-5 or n-7 carbon, i.e rumelenic acid, c9-t11-c15-18:3, whereas di-CLNA have a

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double conjugated double-bond system at the n-5/n-7 or n-8/n-10 carbons, i.e α-eleostaric acid c9-t11-t13-18:3.

Mono-53

CLNA isomers are produced by biohydrogenation of α-linolenic acid by rumen bacteria [5, 6].

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Because of the worldwide problem of obesity in children and the related health problems such as hypertension and diabetes

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mellitus [7], there is an increased interest in the development of preventive and therapeutic strategies for improving insulin

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resistance [8]. Indeed, using antidiabetic drugs is not appropriate for treating diabetes in children unless there is severe

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glucose intolerance, thereby finding natural strategies such as CLNA isomers is an attractive approach which deserves to be

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studied. Di-CLNA are able to decrease body weight [9] and fat[10, 11], as well as increase insulin sensitivity in rodents [9,

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12, 13]. Some studies also reported that either CLA [14] or n-3 PUFA [15] or the combination of the two [16] could

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improve glucose tolerance. Since a mono-CLNA such as c9-t11-c15-18:3 isomer combines the conjugated double bond

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system of CLA and the n-3 double bond of α-linolenic acid, it is reasonable to speculate that this original fatty acid structure

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may provide similar or even enhanced glucose tolerance than the conjugated or n-3 double bond structure.

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Bioavailability of mono-CLNA was reported to be high in rodents. The metabolism of mono-CLNA has already been

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studied in different animal models excluding pigs [5, 6, 17].

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The objective of the present study was to investigate whether dietary supplementation with mono-CLNA (c9-t11-c15-18:3 +

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c9-t13-c15-18:3) improves glucose metabolism in neonatal piglets. In order to isolate the role of the conjugated

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bond in combination with the n-3 PUFA structure, mono-CLNA group will be compared with three other dietary treatments:

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CLA isomers, non-conjugated n-3 and non-conjugated n-6 PUFAs.

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Methods and materials

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Animals and diets

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Thirty two Yorkshire × Landrace × Duroc piglets (females and castrated males) weaned at 3 weeks of age were separated

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into eight groups of four animals. To control for food intake, an oesophageal gastric tube was installed into all animals as

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previously described by Cortamira et al. [18]. Individual adjoining metabolism cages with plastic floors allowed free

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movement and room temperature was kept at 27°C. At baseline, average body weight was 7.6 ± 0.4 kg. Within each group

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the piglets (from the same litter) were assigned to one of four dietary treatment groups, fed entirely with a commercial diet

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(barley (25%), maize (20%), dried whey (20%), soybean meal (10%), extruded soybean (8%) and plasma protein (5%);

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Table 1) plus either 1% of the caloric intake of the basal diet in the form of one the following lipid emulsion of specific fatty

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acids: (1) synthetic mixture of two mono-conjugated α-linolenic acids (c9-t11-c15-18:3 + c9-t13-c15-18:3; CLNA); (2)

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mixture of two conjugated linoleic acids (c9-t11-18:2 + t10-c12-18:2; CLA); (3) n-3 fatty acids (W3); or (4) n-6 fatty acids

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(W6).

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Mono-CLNA isomers were synthesized by alkali isomerization of α-linolenic acid. Thereafter, CLNA isomers were purified

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by preparative chromatography using a reverse phase column, as previously described by Trottier [19]. The fatty acid

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profile of the four lipid emulsions is shown in Table 2.

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All diets were isoenergetic. The feeding regime was based on daily increment of 7.1 g feed per kg0.75 body weight (g/kg0.75)

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up to the target maximum daily value of 56 g/kg0.75. The daily intake was adjusted three times per week according to

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changes in body weight in order to maintain the weight stable.Basal diet was mixed with water (1:2) and infused with a

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syringe into the stomach via the gastric tube. Daily meals were given at 08:00, 11:30 and 16:00 hours; each representing

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45%, 20% and 35% of the diet caloric intake, respectively. Dietary treatments were given during the morning meal. Before

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the morning meal on day 0 (before attribution of treatments) and on day 15 post-weaning, body weight was measured and

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blood samples were collected via jugular venipuncture as previously described by Matte et al. [20].

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On day 9 of the experimental protocol, a jugular catheter was installed by a non-surgical technique described by Matte et al.

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[21]. All animals were tested for insulin resistance by two glucose tolerance tests: a gastro-enteral (OGTT) and a parenteral

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(IVGTT) test. Briefly, after fasting for 18h, piglets (n=32) were given either an oral (OGTT) or an IV (IVGTT) dose of

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glucose (1.0g/kg BW) over a period of 120 min. Blood samples were collected every 30 min for 240 min (0, 30, 60, 90, 120,

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150, 180, 210 and 240 min) starting after the initial glucose infusion. Blood samples were centrifuged at 3000 rpm for 10

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min at 4°C and plasma was stored at -20°C until glucose, insulin and C-peptide analyses were performed. Two days after

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the first glucose test, the protocol was repeated with the other glucose test using the same animal. All animals were

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sacrificed on day 17. The liver was removed, weighed and samples were stored at -20°C for further analysis. The digestive

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tract, brain, lungs and heart were removed and stored at –20°C until lipid quantification was performed.

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Throughout the experimental protocol animals were cared for according to the recommended code of practice of Agriculture

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Canada [22] and the procedure was approved by the local Animal Care Committee following the guidelines of the Canadian

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Council on Animal Care [23].

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Biochemical analyses

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Plasma glucose was measured by an enzymatic colorimetric assay (GLU GOD-PAP; Roche Diagnostics, Indianapolis, IN,

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USA) whereas insulin (Porcine Insulin RIA Kit PI-12K; Linco Research Inc., St Charles, MI USA) and C-peptide (Porcine

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C-peptide RIA kit PCP-22k; Linco Research Inc.) were assayed by commercial RIA kits. The homeostatic model

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assessment (HOMA2), described by Levy et al. [24] was used to estimate insulin sensitivity (HOMA2-%S) and secretion

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(HOMA2-%B) from baseline plasma parameters measured during OGTT and IVGTT. The area under the curve (AUC) of

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glucose, insulin and C-peptide were calculated using the trapezoidal method [25] between 0 and 210 min. Matsuda’s insulin

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sensitivity whole-body index (ISI) was also calculated from the data generated during the OGTT [26]. An insulin sensitivity

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index (SI) derived from the IVGTT was calculated according to a modified method described by Bergman and colleagues

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[27, 28].

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SI was determined from 120 to 210 min of the IVGTT, i.e. after the end of the infusion to assess the deconvolution of

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glucose with regards to insulin after the glucose peak. Total cholesterol, triglyceride and non-esterified fatty acid (NEFA)

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concentration in plasma at day 1 and day 15 were measured by the service diagnostic of the Faculty of Veterinary Medicine

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at the Université de Montréal (Montreal, QC, Canada). The fatty acid composition of the four diets and liver was

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determined by gas chromatography as previously described by Castellano et al. [29].

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Statistical Analysis

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According to results obtained from similar porcine studies [15], the calculated sample size per group (n = 8) was sufficient

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to detect a difference in insulin sensitivity of at least 15% with a power of 80% and a level of significance of 0.05.

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The data were analyzed by using the MIXED procedure implemented in Statistical Analysis Systems software (version 6.11

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of SAS, Cary, NC, USA) [30] according to a completely randomised design with four treatments (CLNA, CLA, W3, and

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W6) as the main factor. The piglet was considered as the experimental unit. The following model was used:

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Yij = µ + Fi + eij

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where Yij is the dependent variable, µ is the overall mean, Fi is the treatment effect and eij is the residual error. Comparisons

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among treatments were done using the following a priori contrasts (CLNA vs. W3 for CLA properties; CLNA vs. W6 for

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both CLA and n-3 PUFA properties; CLNA vs. CLA for n-3 PUFA properties) using a Dunnett’s correction. All values are

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presented as mean ± SEM and differences are considered significant at p<0.05.

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Results

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Anthropometry and blood lipid parameters

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Body weight of piglets pre-treatment was 7.6 ± 0.4 kg. After 2 weeks of supplementation (day 15), there was no difference

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(p>0.82) between treatments for either body weight (10.2 ± 0.4 kg) or fat content (10.0 ± 0.8 %). Total cholesterol,

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triglyceride and NEFA concentrations in blood plasma for day 1 vs. day 15 were, 5.9 ± 0.8 vs. 2.0 ± 0.1 mmol/l, 0.7 ± 0.1

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vs. 0.3 ± 0.1 mmol/l and 708.3 ± 146.7 vs. 769.6 ± 101.8 μmol/l, respectively. There was no difference (p>0.34) according

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to dietary treatments for these parameters.

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Liver fatty acid profile

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There was no significant difference (p=0.67) in liver weight between dietary treatments. The overall organ weight was 248

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± 14 g. Evaluation of liver fatty acid composition was used as an indicator of whole body fatty acid status modification from

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dietary treatments. Mono-CLNA (c9-t11-c15-18:3 + c9-t13-c15-18:3) liver content, was higher (p<0.001) in the CLNA diet

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than the other diets (0.25 vs. 0.01 g/100g fatty acids, respectively). With regards to n-3 fatty acids, 18:3n-3, 20:3n-3 and

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22:5n-3 were 20 to 40% lower (p<0.001) in CLNA diets compared to the W3 diet. Total n-3 PUFA was also significantly

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lower (p<0.001) in the CLNA diet than the W3 diet (9.27 vs. 10.77 g/100g fatty acids, respectively). In contrast, the

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proportion of arachidonic acid (20:4n-6) was 8% higher in the CLNA group compared to the W3 group, resulting in a

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significantly higher total n-6 PUFA level (CLNA vs. W3, 40.61 vs. 39.52 g/100g fatty acids, respectively).

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Basal plasma glucose, insulin and C-peptide concentration

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Baseline plasma glucose, insulin and C-peptide concentrations were evaluated before the OGTT or IVGTT load of glucose

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(Table 3). A significant treatment effect was detected for fasting insulin concentration on the OGTT day (p=0.03) and on

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calculated insulin sensitivity HOMA-%S but the specific contrast test did not allow discrimination between the CLNA

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group and the other dietary groups (p>0.22). There was no other treatment difference for OGTT and IVGTT (p>0.14; Table

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3).

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Monitoring of glucose, insulin and C-peptide during OGTT and IVGTT

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AUC for glucose, insulin and C-peptide monitored between 0 and 210 min are reported in Table 4. Among the four dietary

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groups, there was no significant difference in glucose, insulin and C-peptide monitoring over the OGTT and the IVGTT.

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Dietary intake did not improve the Matsuda’s ISI (p=0.71) calculated from the OGTT nor the minimal model-derived

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insulin sensitivity calculated from the IVGTT (SI; p=0.51).

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Discussion

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The present study aimed to investigate whether dietary supplementation with mono-CLNA improves body composition and

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glucose tolerance in neonatal piglets.

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This model was chosen because piglets represent 1) an accelerated model of postnatal development to study human neonatal

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nutrition and development [31], 2) a relevant model for insulin resistance [32] and 3) a suitable model for evaluating

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nutritional strategies to enhance glucose tolerance and prevent type 2 diabetes and cardiovascular diseases later in life [33].

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Body composition and blood lipids

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Mono-CLNA might combine anti-obesity properties of CLA, along with those of the α-linolenic acid. There is evidence

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suggesting that CLA decreases body weight, fat accumulation and improves serum lipids in mice [34], rats [35], hamsters

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[36] and humans [37]. Similarly, α-linolenic acid was reported to improve the same biomarkers in hamsters [38] and

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humans [39]. Many studies in rodents [10-12, 40-42] showed that dietary di-CLNA supplementation decreases body

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weight, body fat as well as plasma triglycerides and cholesterol. This study speculates that because mono-CLNA has an

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original structure compared to di-CLNA, this conjugated fatty acid will have improved or equally effective glucose

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tolerance than CLA or α-linolenic acid alone. None of them combined an n-3 PUFA and a CLA structure. However, dietary

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supplementation of piglets with mono-CLNA for 14 days did not improve body weight, body fat nor blood lipid profiles.

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Our findings extend previous studies in rodents which reported that dietary mono-CLNA did not lower body weight [17,

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43]. By analogy, dietary di-CLNA isomers did not lower adipose tissue weight as well as total cholesterol and triglycerides

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in the plasma of animals [12, 17, 42, 44, 45] and humans [46].

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Liver fatty acid composition

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CLNA and CLA concentrations in liver reflected the dietary intake of these fatty acids. This response suggests that a

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week supplementation was sufficient for stabilization of PUFA status within the piglet’s body. Our results are in the line

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with Chartrand et al. [47] who reported that dietary fatty acid content consumed for at least 14 days was proportional to

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plasma fatty acid profiles and remained constant up to study completion at 36 days. Our results also showed that giving

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mono-CLNA to piglets changed the n-3 and n-6 fatty acid balance. More specifically, compared to the W3 diet, feeding

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mono-CLNA for 2 weeks decreased the proportions of 18:3n-3, 20:3n-3, 20:5n-3, 22:5n-3 together with an increase in

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20:4n-6 and total n-6 PUFA content. Since a previous study in mice reported that n-3 PUFA chronic depletion in the liver

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led to the development of hepatic insulin resistance over a 3 month period [48], further studies need to be carried out for an

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extended period of time in pigs fed CLNA with additional health indicators (blood biochemical, other tissue lipid profile,

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etc) in order to better assess the safety aspects of consuming dietary mono-CLNA isomers.

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Glucose tolerance

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One of our hypotheses was that combining a conjugated and an n-3 PUFA structure in one fatty acid, like mono-CLNA,

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would improve insulin sensitivity considering that dietary CLA seems to lower insulinemia in rats [49] and α-linolenic acid

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intake seems to reduce insulin resistance in rats [50] and in humans [51].

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Our results suggest a significant treatment effect for fasting insulin concentration and insulin sensitivity on the OGTT day

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(p=0.03). However, we consider that this result is unlikely of biological significance because specific a priori contrasts

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indicate that there is no treatment effect of mono-CLNA diet compared to the three other dietary groups. Moreover, these

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differences were not confirmed at the day of IVGTT or during the two glucose tolerance tests. One possible explanation

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could be a higher insulin secretion generated by environmental/psychological stresses during the first experiment, i.e.

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human presence, handling, noise, etc [52, 53]. Also, no treatment effect was seen on C-peptide, a good indicator of

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endogenous insulin secretion [54], either for OGTT or IVGTT.

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In regards to the different indexes of insulin sensitivity (HOMA2-%S, ISIMatsuda and SI) and insulin secretion (Insulin and

C-211

peptide levels, AUCs and HOMA2-%B), none were improved by ingestion of mono-CLNA for 2 weeks.

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Results on a closer structural analog to mono-CLNA such as di-CLNA showed some inconsistency. Indeed, although

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several studies reported that dietary supplementation with di-CLNA isomers can decrease type 2 diabetes risk [12] and

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improve glucose tolerance [9, 55] in mice, others reported an increase in insulin resistance (HOMA-IR index) in rats [44]

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similar to what is reported in mice [56, 57], pigs [58], and humans [59]. Moreover, most of the studies using n-3 PUFA

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supplement in humans failed to improve insulin sensitivity [60, 61].

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Limitations of the present study

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The present study extends previous findings [9, 40, 44, 55] using a neonatal pig model and randomised experimental design

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to compare mono-CLNA diet vs. three other dietary treatments (CLA, W3, and W6). Nevertheless, it has some limitations

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including the duration of the supplementation and the composition of the CLA diet, since we used a mixture of two isomers:

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c9-t11-18:3 + t10-c12-18:3. Even if most previous studies have used a CLA isomer mixture, recent findings show that

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purified CLA isomers could have opposite actions on glucose tolerance, with t10-c12-18:3 reducing insulin sensitivity and

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c9-t11-18:3 enhancing insulin tolerance [3]. Mono-CLNA was also a mixture of two isomers and this is mostly because it is

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not possible to cost effectively separate the two CLNA isomers for generating high doses of single CLNA isomers for

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feeding animals. Therefore, a direct comparison between CLA and CLNA diets based only on the chemical structure is

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limited.

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Conclusions

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This study showed that mono-CLNA, combining conjugated and an n-3 double-bound structure, did not provide additive

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improvement for body composition, glucose tolerance or blood lipid profile in the neonatal piglet model supplemented for a

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period of 2 weeks. Conversely, mono-CLNA decreased total n-3 PUFA in liver, a finding which merits consideration in

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regards to neonatal development and safety.

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Acknowledgments

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The authors are grateful to M. Guillette for her invaluable technical assistance; to Mélanie Turcotte and the animal care

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team under supervision of D. Morrissette; and to S. Methot for his help with statistical analysis. The authors are also

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grateful to Fonds u b cois de echerche sur la Nature et les Technologies (FQRNT) and Naturia Inc (Sherbrooke,

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Canada) for a Ph.D scholarship to M. P at the time where the experiment was carried-out and to Fonds de recherche

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Québec-santé for a current Junior 1 salary award. Mono-CLNA were generously provided by Naturia Inc. (Sherbrooke, QC,

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Canada). The financial support was provided by Naturia Inc and the Mathing Investment Initiative of Agriculture and

Agri-242

Food Canada. None of the authors has any financial conflict of interest.

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Table 1: Composition of basal diet

395

Ingredients Calculated concentration

Digestible energy (MJ/kg) 14.98 Total protein (%) 19.94 Crude fibre (%) 1.90 Fat (%) 7.49 Lysine (%) 1.40 Methionine (%) 0.43 Tryptophan (%) 0.24 Calcium (%) 0.80 Phosphorus (%) 0.70

Provided (per kg basal diet): Mn, 40 mg; Zn, 2935 mg; Fe, 299 mg; Cu, 19 mg; I, 2 mg; Se 297 μg; vitamin A, 4.9 mg; vitamin D, 37.5 μg; vitamin E, 66.8 mg; menadione, ; thiamin, 2.7 mg; riboflavin, 8.7 mg; niacin, 31 mg; panthothenic acid 21.2 mg; folic acid, 0.7 mg; pyridoxine, 2.6 mg; biotin, 120 μg; vitamin B12, 25.1 μg; choline, 303 mg

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15

Table 2: Analytical fatty acid composition (g/100 g fatty acids) of lipid emulsions added to the dietary treatments

397

Fatty acid CLNA CLA W3 W6

16:0 6.37 6.04 6.10 5.97 16:1n-7 0.05 0.04 0.07 0.08 18:0 3.23 3.39 3.60 3.63 18:1n-9 22.40 21.61 23.39 22.89 18:1n-7 0.01 0.04 0.06 0.05 18:2n-6 15.72 19.49 18.58 53.08 18:3n-3 12.65 13.24 46.99 12.88 18:3n-6 0.19 0.08 0.21 0.10 20:0 0.06 0.14 0.14 0.23 20:1n-9 0.02 0.01 0.02 0.08 20:2n-6 1.72 1.50 0.06 0.07 20:3n-3 0.40 0.04 0.08 0.03 20:3n-6 1.13 0.03 0.04 0.03 20:4n-6 0.10 0.01 0.01 0.01 20:5-n-3 0.62 0.02 0.01 0.01 22:1n-9 0.66 0.04 0.08 0.03 22:5n-3 0.11 0.03 0.12 0.03 22:6n-3 0.05 0.04 0.03 0.03 24:0 0.84 0.07 0.06 0.12 24:1n-9 0.59 0.12 0.01 0.03 c9-t11-18:2 0.87 16.82 0.03 0.05 t10-c12-18:2 1.70 16.77 0.02 0.02 c9-t11-c15-18:3 + c9-t13-c15-18:3 30.33 0.25 0.16 0.46 Total conjugated 32.61 33.50 0.24 0.53 SFA 10.24 9.63 9.92 9.95 MUFA 23.86 21.92 23.90 23.34 PUFA 65.87 68.45 66.23 66.81 n-3:n-6 ratio 2.06 0.25 2.50 0.25

SFA = total saturated fatty acids; MUFA = total monounsaturated fatty acid; PUFA = total polyunsaturated fatty acids; n-3:n-6 ratio = n-3 PUFA to n-6 PUFA ratio

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16

Table 3: Basal plasma concentrations of glucose, insulin and C-peptide during OGTT or IVGTT according to the dietary

399

treatments

400

Diet

Index CLNA CLA W3 W6 SEM P-value

OGTT Glucose (mmol/l) 5.6 5.4 5.5 5.8 0.2 0.74 Insulin (pmol/l) 58.8 50.3 63.9 62.6 5.9 0.03 C-peptide (pmol/l) 53.0 64.4 68.3 49.9 7.9 0.27 HOMA2-%Sa 94.6 106.6 87.0 89.8 9.0 0.05* HOMA2-%Ba 82.0 79.1 89.3 80.7 6.7 0.57 IVGTT Glucose (mmol/l) 5.8 5.3 5.3 5.6 0.3 0.26 Insulin (pmol/l) 57.3 64.3 55.2 58.9 6.9 0.62 C-peptide (pmol/l) 43.9 58.8 57.3 66.9 9.0 0.31 HOMA2-%Sa 88.5 102.3 98.8 92.7 12.5 0.76 HOMA2-%Ba 82.8 96.6 88.2 81.5 7.4 0.14

W3 = omega-3 fatty acids diet; W6 = omega-6 fatty acids diet; CLNA = conjugated alpha-linolenic acids diet; CLA = linoleic acids diet; OGTT=oral glucose tolerance test; IVGTT = intravenous glucose tolerance test.

aCalculated insulin sensitivity (HOMA2-%S) and β-cell function (HOMA2-%B) based on homeostatic model assessment [24].

*Specific contrasts p-values for CLNA vs. CLA, CLNA vs. W3 and CLNA vs. W6 were 0.22, 0.53 and 0.81, respectively.

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17

Table 4: Plasma glucose, insulin and C-peptide responses in OGTT and IVGTT

402

Diet

Index CLNA CLA W3 W6 SEM P-value

OGTT Glucose (mmol × min/l)a 23.3 22.6 22.2 22.9 0.6 0.56 Insuline (nmol × min/l)a 478.3 441.1 453.0 426.4 36.4 0.70 C-peptide (nmol × min/l)a 618.5 550.5 573.8 563.8 52.6 0.68 ISI (0, 210 min)b 7.7 8.7 7.7 7.9 0.6 0.21 IVGTT Glucose (mmol × min/l)a 28.5 27.6 28.2 27.5 0.9 0.81 Insuline (nmol × min/l)a 517.5 565.9 530.9 484.6 36.8 0.42 C-peptide (nmol × min/l)a 706.1 791.8 744.3 697.8 54.58 0.55 SI (120, 210 min)c 5.0 4.5 4.7 5.4 0.68 0.51

W3 = omega-3 fatty acids diet; W6 = omega-6 fatty acids diet; CLNA = conjugated alpha-linolenic acids diet; CLA = linoleic acids diet; OGTT = oral glucose tolerance test; IVGTT = intravenous glucose tolerance test;

aValues are AUC from 0 to 210 min during OGTT or IVGTT.

b

Insulin sensitivity index (ISI) [26] calculated as follow: ISI= (Glubasal × Insbasal × Glumean × Insmean)0.5. cMinimal model-derived insulin sensitivity index (SI) based on MINMOD Millennium [28].

Figure

Table 1: Composition of basal diet
Table 2: Analytical fatty acid composition (g/100 g fatty acids) of lipid emulsions added to the dietary treatments
Table 3: Basal plasma concentrations of glucose, insulin and C-peptide during OGTT or IVGTT according to the dietary
Table 4: Plasma glucose, insulin and C-peptide responses in OGTT and IVGTT

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